The last two decades witnessed a huge increase in our understanding of outer hair cell (OHC) structure and function. We know, for example, that cochlear amplification is associated with OHC fast motility, which is driven by forces generated in the plasma membrane by conformational changes in thousands of copies of the integral-membrane protein prestin. We know that these forces are harnessed and oriented parallel to the OHC's major axis by the cortical cytoskeleton, a two-dimensional anisotropic network of actin filaments cross-linked by spectrin that underlies the entire OHC lateral plasma membrane, and we know that that is possible because the plasma membrane is linked to cytoskeletal actin filaments by thousands of structural "pillars". We still don't know, however, the nature of these pillars. We don't know how they work, or whether they have any role in the regulation of the prestin-generated force. We don't know either the molecular mechanisms that control OHC length and motility by regulating cytoskeletal organization and its pillar-mediated connection to the plasma membrane. The goal of this proposal is to fill these gaps in our knowledge of OHC's structure and function, a crucial step in our quest to understand cochlear function, malfunctions that involve changes in OHC shape, and protective mechanisms in response to over stimulation, by addressing the following Specific Aims: 1) Demonstrate that proteins GLUT5, Dematin and Adducin are major components of OHC pillars, 2) Determine the role of actin polymerization and depolymerization induced by profilin- and cofilin-mediated signals in the regulation of OHC length and motility, and 3) Determine the role of the RhoA/ROCK-PKC pathway in the modulation of OHC length and motility. Structural and/or functional damage of the organ of Corti is the major cause of sensorineural hearing loss afflicting millions of people around the world. The exquisite architecture of the organ of Corti is uniquely adapted to support an enormous range of input sound pressures, and outer hair cells (OHCs) are unique in having their body length directly influencing important aspects of the micromechanics of this organ. For example, minute changes in OHC length can dynamically adjust the operating point of the mechano-sensory apparatus hosted in the cell stereocilia and/or the local resonance of the basilar membrane. Changes in OHC length, known as "OHC motility", are thought to be essential for cochlear amplification. Despite their importance, many gaps exist in our knowledge of the structure and function of OHCs. Filling these gaps would be an important step towards a better understanding of cochlear function, malfunctions that involve changes in OHC shape, and protective mechanisms activated in response to overstimulation. We are confident that accomplishing the aims of the present proposal will provide essential information about the structure of OHCs and the role of the cytoskeleton in the regulation of OHC length and motility, as well as critical insights into the basic mechanisms of both normal human hearing and deafness.